Anti-Inflammatory Effects of Spirodela polyrhiza (L.) SCHLEID. Extract on Contact Dermatitis in Mice—Its Active Compounds and Molecular Targets

Spirodela polyrhiza (L.) SCHLEID. has been used to treat epidemic fever, dysuria, and various skin ailments, such as measles eruptions, eczema, and pruritus, in China, Japan, and Korea. In this study, the active compounds in S. polyrhiza and their target genes were identified by network-based analysis. Moreover, the study evaluated the effects of a 70% ethanolic extract of S. polyrhiza (EESP) on skin lesions, histopathological changes, inflammatory cytokines, and chemokines in mice with contact dermatitis (CD) induced by 1-fluoro-2,4-dinitrobenzene (DNFB), and examined the inhibitory effects of EESP on mitogen-activated protein kinase (MAPK) signalling pathways. In our results, 14 active compounds and 29 CD-related target genes were identified. Among them, tumour necrosis factor (TNF) and interleukin 6 (IL-6) were identified as hub genes, and luteolin and apigenin showed a strong binding affinity with TNF (<−8 kcal/mol) and IL-6 (<−6 kcal/mol). Our in vivo studies showed that topical EESP ameliorated DNFB-induced skin lesions and histopathological abnormalities, and reduced the levels of TNF-α, interferon (IFN)-ɣ, IL-6, and monocyte chemotactic protein (MCP)-1 in inflamed tissues. In conclusion, our findings suggest the potential for dermatological applications of S. polyrhiza and suggest that its anti-dermatitis action is related to the inhibition of TNF and IL-6 by luteolin and luteolin glycosides.


Introduction
Contact dermatitis (CD) has a long history, dating back to ancient Egypt, as evidenced by its appearance on papyrus scrolls.Documentation of CD has been found in many different cultures and countries, not only in Europe but also in China and India [1].At present, CD can have a significant impact on quality of life, and 20.1% of the general population have contact allergies to certain substances [2].Occupational skin diseases (OSD) contribute to around 25% of all work-related lost days, and CD is responsible for 70-90% of OSD cases [3,4].CD causes itching and blistered, dry, cracked skin, and can cause lighter skin to redden and darker skin to become dark brown, purple, or grey [5].
Corticosteroids are considered the gold-standard for managing acute CD flare-ups and, when used appropriately, are generally safe for short-term use.However, the long-term use or misuse of potent corticosteroids can lead to adverse effects such as skin thinning, discoloration, and increased infection risk.In instances involving unavoidable and repetitive use, such as OCD, this can pose a greater challenge.Thus, novel therapeutic agents are required, and complementary and alternative medicines based on natural substances offer relatively safe alternatives [6,7].
Spirodela polyrhiza (L.) S CHLEID ., commonly known as duckweed, is a species of aquatic flowering plant belonging to the family Araceae.Duckweeds are small, free-floating plants found in ponds, lakes, slow-moving streams, and other freshwater bodies worldwide.In East Asian literature, duckweed is often used as a metaphor for a fleeting or wandering life.Duckweeds, including this species, have been well-studied because of their ability to remove pollutants from wastewater, which makes them a promising tool for water treatment.Additionally, they can be used as a food source for livestock, poultry, and fish due to their high protein contents [8].
In traditional East Asian medicine, whole S. polyrhiza is a diaphoretic with a cool property and is used to induce sweating, reduce fever, and as a diuretic and detoxifier.Traditional medicine experts in China, Japan, and Korea have used this plant to treat epidemic fever, dysuria, and skin ailments, such as measles eruption, eczema, and pruritus [9].Furthermore, S. polyrhiza has been reported to have anti-inflammatory [10] and anti-allergic effects [11][12][13].
We aimed to investigate whether the candidate plant found in traditional Korean medicine has therapeutic effects on CD and to explore its active compounds and associated mechanisms.In this study, active compounds of S. polyrhiza and their targets were identified using network pharmacological analysis.In addition, the anti-inflammatory efficacy of a 70% ethanolic extract of whole S. polyrhiza (EESP) was investigated in a murine model of CD.

Result 2.1. Fourteen Active Compounds Were Found in S. polyrhiza
In total, 73 compounds were found in the TCMSP, ETCM and TCMBank databases (Supplementary Data S1, Table S1), and target gene list of 730 was obtained (Supplementary Data S2, Table S2).Of these, 23 compounds had CD-related gene targets as determined by the DisGeNET database and 14 compounds ((+)-armepavine, (R)-N-methylcoclaurine, 2-hexadecenoic acid, anonaine, apigenin, butylated hydroxytoluene, D-ascorbic acid, higenamine, isoliensinine, luteolin, nornuciferine, nuciferine, palmitic acid and roemerine) satisfied the ADME criteria.Among them, six compounds, including apigenin, D-ascorbic acid and luteolin, had a DrugBank (https://go.drugbank.com/(accessed on 16 August 2023)) accession number (Table 1).Corticosteroids are considered the gold-standard for managing acute CD flare-ups and, when used appropriately, are generally safe for short-term use.However, the longterm use or misuse of potent corticosteroids can lead to adverse effects such as skin thinning, discoloration, and increased infection risk.In instances involving unavoidable and repetitive use, such as OCD, this can pose a greater challenge.Thus, novel therapeutic agents are required, and complementary and alternative medicines based on natural substances offer relatively safe alternatives [6,7].
Spirodela polyrhiza (L.) SCHLEID., commonly known as duckweed, is a species of aquatic flowering plant belonging to the family Araceae.Duckweeds are small, free-floating plants found in ponds, lakes, slow-moving streams, and other freshwater bodies worldwide.In East Asian literature, duckweed is often used as a metaphor for a fleeting or wandering life.Duckweeds, including this species, have been well-studied because of their ability to remove pollutants from wastewater, which makes them a promising tool for water treatment.Additionally, they can be used as a food source for livestock, poultry, and fish due to their high protein contents [8].
In traditional East Asian medicine, whole S. polyrhiza is a diaphoretic with a cool property and is used to induce sweating, reduce fever, and as a diuretic and detoxifier.Traditional medicine experts in China, Japan, and Korea have used this plant to treat epidemic fever, dysuria, and skin ailments, such as measles eruption, eczema, and pruritus [9].Furthermore, S. polyrhiza has been reported to have anti-inflammatory [10] and antiallergic effects [11][12][13].
We aimed to investigate whether the candidate plant found in traditional Korean medicine has therapeutic effects on CD and to explore its active compounds and associated mechanisms.In this study, active compounds of S. polyrhiza and their targets were identified using network pharmacological analysis.In addition, the anti-inflammatory efficacy of a 70% ethanolic extract of whole S. polyrhiza (EESP) was investigated in a murine model of CD.

Fourteen Active Compounds and Twenty-One CD-Related Targets Were Closely Associated
The results of identifying target genes for 14 active compounds revealed that, among the 29 CD-related genes shown in Figure 1, 21 genes were identified.Correlations between 14 active compounds and 21 genes were analysed using Cytoscape 3.91.The results

Fourteen Active Compounds and Twenty-One CD-Related Targets Were Closely Associated
The results of identifying target genes for 14 active compounds revealed that, among the 29 CD-related genes shown in Figure 1, 21 genes were identified.Correlations between 14 active compounds and 21 genes were analysed using Cytoscape 3.91.The results showed that apigenin interacted with 11 disease targets, while D-ascorbic acid (vitamin C) and luteolin were associated with 10 and 8, respectively.In addition, TNF was associated with five active compounds, including apigenin, D-ascorbic acid, and luteolin.IL6 was connected to D-ascorbic acid and luteolin.IFNG was correlated with apigenin and luteolin.Lastly, monocyte chemotactic protein-1 (MCP-1, (CCL2)) was found to be associated with D-ascorbic acid (Figure 2).showed that apigenin interacted with 11 disease targets, while D-ascorbic acid (vitamin C) and luteolin were associated with 10 and 8, respectively.In addition, TNF was associated with five active compounds, including apigenin, D-ascorbic acid, and luteolin.IL6 was connected to D-ascorbic acid and luteolin.IFNG was correlated with apigenin and luteolin.Lastly, monocyte chemotactic protein-1 (MCP-1, (CCL2)) was found to be associated with D-ascorbic acid (Figure 2).

Eleven and Eight Active Compounds Showed Strong Binding Affinity to TNF or IL-6, Rrespectively
Binding affinities between TNF or IL-6 and the 14 active compounds were predicted by molecular docking analysis (Table 2).Eleven compounds showed strong binding affinity with TNF with binding energies of <−7 kcal/mol, and eight compounds showed strong binding affinity with IL-6 with binding energies of <−6 kcal/mol.The binding modes of the active compounds and residues of TNF or IL-6 are shown in Figure 3A,B.

Eleven and Eight Active Compounds Showed Strong Binding Affinity to TNF or IL-6, Rrespectively
Binding affinities between TNF or IL-6 and the 14 active compounds were predicted by molecular docking analysis (Table 2).Eleven compounds showed strong binding affinity with TNF with binding energies of <−7 kcal/mol, and eight compounds showed strong binding affinity with IL-6 with binding energies of <−6 kcal/mol.The binding modes of the active compounds and residues of TNF or IL-6 are shown in Figure 3A,B.

EESP Ameliorated Skin Lesions and Inhibited Skin Thickening in CD Mice
The repeated topical application of 1-fluoro-2,4-dinitrobenzene (DNFB) resulted in the development of skin lesions characteristic of CD that exhibited skin scaling, excoriation, erythema, and roughness (Figure 5A,B).Topical EESP administered for 6 days provided relief from these symptoms, as depicted in Figure 5A.Additionally, when treated with EESP (150 or 500 µg/day), skin surface scores were significantly reduced (Figure 5B); notably, mean skin weight was considerably greater in the CTL group than in the NOR group, whereas the application of EESP effectively prevented skin weight increases (Figure 5C).The application of DNFB resulted in a significant increase in melanin and erythema indices in the CTL group; when EESP was applied at 500 µg/day, there was a notable reduction in melanin indices, as shown in Figure 5D.Furthermore, EESP application at 150 or 500 µg/day significantly reduced erythema indices (Figure 5E).

EESP Ameliorated Skin Lesions and Inhibited Skin Thickening in CD Mice
The repeated topical application of 1-fluoro-2,4-dinitrobenzene (DNFB) resulted in the development of skin lesions characteristic of CD that exhibited skin scaling, excoriation, erythema, and roughness (Figure 5A,B).Topical EESP administered for 6 days provided relief from these symptoms, as depicted in Figure 5A.Additionally, when treated with EESP (150 or 500 µg/day), skin surface scores were significantly reduced (Figure 5B); notably, mean skin weight was considerably greater in the CTL group than in the NOR group, whereas the application of EESP effectively prevented skin weight increases (Figure 5C).The application of DNFB resulted in a significant increase in melanin and erythema indices in the CTL group; when EESP was applied at 500 µg/day, there was a notable reduction in melanin indices, as shown in Figure 5D.Furthermore, EESP application at 150 or 500 µg/day significantly reduced erythema indices (Figure 5E).

EESP Prevented DNFB-Induced Histopathological Abnormalities
The repeated application of DNFB resulted in notable epidermal hyperplasia, hyperkeratosis, epidermis destruction, and immune cell infiltration (Figure 6A).Treatment with

EESP Effectively Reduced DNFB-Induced Increases in TNF-α, IFN-γ, IL-6, and MCP-1 Levels in inflamed tissues
TNF-α, IFN-γ, IL-6, and MCP-1 levels were significantly elevated in the DNFBtreated skins of mice in the CTL group.When EESP was topically applied at a dosage of 500 µg/day, it effectively reduced these DNFB-induced increases in inflammatory cytokine and chemokine levels.EESP at all concentrations administered significantly lowered TNF-α levels and, at 150 and 500 µg/day, EESP also significantly lowered IL-6 and MCP-1 levels (Figure 7).

EESP Effectively Reduced DNFB-Induced Increases in TNF-α, IFN-γ, IL-6, and MCP-1 Levels in Inflamed Tissues
TNF-α, IFN-γ, IL-6, and MCP-1 levels were significantly elevated in the DNFBtreated skins of mice in the CTL group.When EESP was topically applied at a dosage of 500 µg/day, it effectively reduced these DNFB-induced increases in inflammatory cytokine and chemokine levels.EESP at all concentrations administered significantly lowered TNF-α levels and, at 150 and 500 µg/day, EESP also significantly lowered IL-6 and MCP-1 levels (Figure 7).

Discussion
In the network pharmacologic analysis, we identified 14 active compounds and 29 target genes in S. polyrhiza.Among them, IL6 and TNF were identified as hub genes.Among the 14 active compounds, apigenin and luteolin showed a strong binding affinity with both TNF and IL-6.Our in vivo studies showed that EESP ameliorated skin lesions and histopathological abnormalities in CD mice.In addition, EESP effectively reduced DNFBinduced increases in TNF-α, IFN-γ, IL-6, and MCP-1 levels in inflamed tissues.These results imply that S. polyrhiza can exert an anti-inflammatory effect in CD and that the therapeutic mechanism is related to the inhibition of cytokines such as TNF and IL-6.
The anti-inflammatory effects of S. polyrhiza have been established in several previous studies [10,13].The fact that it effectively reduces the increase in TNF-α and IL-6 induced by lipopolysaccharide (LPS) stimulation in RAW264.7 cells [10], along with the significant decrease in TNF-α and IL-6 observed in an atopic dermatitis animal model [13], consistently aligns with the findings of our study.
Using a network pharmacologic approach, we identified 14 active compounds, including apigenin, D-ascorbic acid, and luteolin (Table 1), as well as 29 target genes, which included TNF, IL6, IL1B and IL4 in S. polyrhiza.These may serve as markers of the antiinflammatory effect of S. polyrhiza on the skin.An analysis of correlations between the 29 target genes using the STRING platform and the cytoHubba plugin revealed that IL6 and TNF were identified as hub genes (Figure 1 and Supplementary Data S4).This indicates that TNF and IL6 should be given priority consideration for research on the anti-inflammatory effects of S. polyrhiza, owing to their high centrality.
For the identification of enriched transcriptomic signatures, analysis was conducted by using Enrichr website.The enrichment analysis of RNAseq automatic Gene Expression Omnibus (GEO) signatures revealed that active compounds of S. polyrhiza (730 target genes) were associated with mouse down gene set including Atopic Dermatitis Nestin-Cre IKK2 (GSE109936) [14] (Supplementary Data S6, Figure S2).
Among the 14 active compounds, apigenin, D-ascorbic acid and luteolin were directly linked to TNF (Figure 2).Among active compounds, apigenin, luteolin and butylated hydroxytoluene were linked to both TNF and HMOX1, eight compounds, except for the six compounds related to OPRM1, were linked to antioxidant or anti-inflammatory factors.These results mean that they can have antioxidant and anti-inflammatory effects, and that antioxidant and anti-inflammatory actions will play an important role in the therapeutic mechanism of S. polyrhiza.
Interestingly, OPRM1 and six active compounds did not correlate with other target genes or compounds (Figure 2).In addition, there was no connection between OPRM1 and other target genes in the protein-protein interaction (PPI) analysis (Figure 1).These results suggest the possibility of dividing the compound groups of S. polyrhiza related to the treatment of CD into two clusters: the itching improvement group (OPRM1-related 6 compounds) [15] and the antioxidant and anti-inflammatory group.
As shown in Table 2 and Figure 3, apigenin and luteolin could stably bind to TNF and IL-6.As apigenin is directly linked to TNF and luteolin is directly linked to both TNF and IL-6 in Figure 2, this result can be interpreted as the possibility that these two compounds bind to TNF or IL-6 and inhibit its function.Further research in this area will be needed.On the other hand, D-ascorbic acid had a binding affinity greater than -6 (Table 2) despite being directly linked to TNF and IL-6 (Figure 2).This seems to be because D-ascorbic acid indirectly inhibits TNF or IL-6 through its antioxidant effect.
As shown in Figure 4, apigenin, apigenin-7-glucoside, luteolin and luteolin-7-glucoside were detected in EESP by HPLC, and the two peaks appearing before these four main peaks were assigned to luteolin-8-glucoside and apigenin-8-glucoside, respectively [16].Apigenin and luteolin are standard substances and commonly used active compounds for the authentication of S. polyrhiza [11,17].The S. polyrhiza extract used in this study contained a relatively large amount of luteolin compared to apigenin (Figure 4).This result implies that luteolin was mainly involved in the anti-inflammatory activity of the S. polyrhiza extract used in this experiment.
In the present study, we found that the repeated application of EESP significantly improved scaling, erythema, and pigmentation, and reduced skin weights in our DNFBinduced mouse model (Figure 5).In addition, EESP also inhibited epidermal hyperplasia, immune cell infiltration, and hyperkeratosis (Figure 6).These observations suggest that topical EESP can suppress excessive epidermal cell growth, inhibit skin-thickening, and reduce abnormal keratinization.In addition, the skin-lightening effect that was observed raises the possibility that EESP has potential use as a skin-whitening agent and well-known inhibitors of melanogenesis, such as luteolin [18], may be involved in this action of EESP.
TNF-α and IFN-γ play crucial roles in the initiation and amplification of inflammatory responses, and both can activate keratinocytes, promote the release of pro-inflammatory mediators and disrupt skin barrier function [19], which causes hyperplasia, erosion, and hyperkeratosis in the epidermis [20].IL-6 induces the production of other pro-inflammatory cytokines, such as IL-1β and TNF-α, by keratinocytes and modulates the production and distribution of key proteins involved in skin barrier integrity, such as filaggrin and tight junction proteins [21].MCP-1 (also known as CCL2) functions as a chemoattractant for various immune cells, including monocytes, T cells, neutrophils, and dendritic cells [22].In addition, IL-6, TNF-α, and IFN-γ act in concert to promote immune cell recruitment and inflammation by enhancing the expression of adhesion molecules in keratinocytes [19][20][21] and accelerating immune cell infiltration into inflamed tissues, thus amplifying the inflammatory response.
In the present study, EESP effectively lowered TNF-α, IFN-G, IL-6, and MCP-1 levels in the inflamed skin tissues of CD mice (Figure 7) and inhibited skin-thickening and epidermal hyperplasia (Figures 5 and 6).Our results indicate EESP suppresses skinthickening by inhibiting pro-inflammatory cytokine and chemokine production, preventing keratinocyte accumulation, and thus suppressing epidermal hyperplasia.In addition, EESP significantly suppressed immune cell infiltration (Figure 6C), which we presume was due to its inhibition of pro-inflammatory cytokine and chemokine production.Collectively, these findings suggest that EESP can suppress the production of TNF-α, IFN-γ, IL-6, and MCP-1 and consequently inhibit epidermal hyperplasia, hyperkeratosis, and immune cell infiltration, which ameliorate the skin symptoms of CD and suppress skin-thickening.Considering the results in Figure 2, which show a connection between apigenin, D-ascorbic acid, and luteolin with TNF, IFNG, IL6, and CCL2 (MCP-1), it seems that these three main active compounds are associated with inhibitory effects on the production of cytokines and chemokine.
MAPK signalling plays an important role in the pathogenesis and progression of CD and cytokine secretion, including IL-6 and TNF-α [23,24].Our results show that EESP prevented the DNFB-induced phosphorylation of ERK (Figure 8).In addition, Lee et al. reported that ethanol extracts of S. polyrhiza inhibited the JNK, ERK and p38 MAPK pathways in an animal model of atopic dermatitis [13].These results imply that the anti-inflammatory mechanism initiated by EESP is involved in the regulation of cytokine production through inhibition of the MAPK pathway.
Luteolin can attenuate adverse photobiological effects on the skin and has antioxidative and anti-inflammatory effects on keratinocytes, fibroblasts, and several types of immune cells.In addition, luteolin can suppress pro-inflammatory mediators and regulate various signalling pathways [25].We found that luteolin was correlated with 8 of the 29 selected disease target genes, including TNF, IFNG, and IL6 (Figure 2).In addition, luteolin showed strong binding affinities with both TNF and IL-6 (Table 2 and Figure 3).These findings indicate that luteolin underpins the anti-dermatitis effect of EESP and offers a starting point for the development of topical and systemic agents for inflammatory skin diseases.
We found that topical DEX administration suppressed weight gain and decreased spleen/body weight ratios, which is considered an indicator of general immune sup-pression [26], in CD mice, while EESP had no effect (Supplementary Data S7, Figure S3).These findings imply that the anti-inflammatory mechanism of EESP differs from that of corticosteroids, especially in terms of systemic immune suppression.
As shown in Figure 5D,E, DEX did not exhibit any improvement in skin color changes induced by DNFB.This phenomenon is often observed in experimental models of CD and commonly coincides with skin atrophy.Furthermore, there are instances where DEX fails to ameliorate surface symptoms on the skin [26][27][28].The therapeutic mechanism of corticosteroids in eczema primarily involves their anti-inflammatory effects, which are achieved through immunosuppression rather than direct enhancement of the skin barrier function.On the other hand, it is worth noting that corticosteroid usage can lead to skin atrophy, potentially resulting in increased redness or darkening of the skin due to thinning.For this reason, while corticosteroids effectively suppress inflammation, the development of complementary agents aiming to restore skin integrity and improve the skin barrier function appears to be essential.
We did not perform pharmacokinetic studies.Therefore, there is no basis to determine whether the effect of EESP is local or systemic.Since EESP is applied to the skin, it is necessary to study how certain compounds are absorbed through the skin, how they are distributed, and how they are metabolized.In addition, because we have not checked all the compounds in EESP, it is hard to know what compounds are in the sample, how much of the sample they are present in, and what they do.Additionally, compounds for which a PubChem CID could not be identified were excluded from the analysis.For these compounds, the swissADME database cannot be used because the canonical SMILES is unknown.

Network Analysis
Relationships between disease-related genes were analysed using STRING platform (https://string-db.org/(accessed on 16 August 2023)).The network edge was displayed as a confidence level, and the minimum required interaction score was set to 0.7 (high confidence) or higher.The cytoHubba plugin was used for the identification of hub genes from the PPI analysis [33].Cytoscape 3.9.1 (https://cytoscape.org/(accessed on 16 August 2023)) was used to confirm compound-target networks [34].For the identification of enriched transcriptomic signatures, an analysis was carried out using Enrichr website (https://maayanlab.cloud/Enrichr/(accessed on 18 August 2023)).

Docking Analysis
Docking analysis was performed as described previously [28], and the binding affinities of active compounds and target proteins were determined using AutoDock Vina [35].The structures of the two proteins (PDB file) were obtained from the protein data bank (PDB, https://www.rcsb.org/(accessed on 15 June 2023)) database, and the structures of the 14 active compounds were obtained from the PubChem (https://pubchem.ncbi.nlm.nih.gov/(accessed on 15 June 2023)) database.Structures obtained for docking analysis were edited and visualized using AutoDockTools (ver 1.5.7,Molecular Graphics Laboratory, Greensboro, NC, USA) and PyMOL (The PyMOL Molecular Graphics System ver.2.5.5, Schrödinger, New York, NY, USA) programs.The analysis process is summarized in Supplementary Data (Supplementary Data S8, Figure S4).EESP was dissolved in methanol at 10 mg/mL.Apigenin, apigenin-7-glucoside, luteolin, and luteolin-7-glucoside were dissolved in methanol at 0.5 mg/mL.EESP and the standard mixture were filtered through a 0.20 µm Syringe Filter (Bio FACT TM, Daejeon, Republic of Korea) before HPLC.

Chromatographic Conditions
HPLC was performed using an Agilent series 1100 instrument (Agilent, Waldbronn, Germany) equipped with a quaternary pump, a variable wavelength detector, and an autosampler.Samples were separated on a Capcell Pak C18 column (5 µm, 4.6 × 250 mm, Shiseido, Tokyo, Japan).The mobile phase consisted of mixtures of water containing 0.5% (v/v) formic acid (A) or 100% methanol (B).The following gradient elution program was used: 30-40% B for 0-20 min, 40-53% B for 20-30 min, 53-90% B for 30-40 min, and 90% B for 40-45 min.The unit was operated at a flow rate of 1.0 mL/min and a column temperature of 35 • C, and the UV detector was set at 254 nm.Data were processed using Agilent LC B.02.01 ChemStation software.

The Anti-Inflammatory Effects of EESP in
Mice with CD 4.3.1.Preparation of S. polyrhiza Extract S. polyrhiza was purchased from the Kwangmyungdang Company (Ulsan, Republic of Korea), and a specimen was deposited in the School of Korean Medicine, Pusan National University (Voucher no.MS2017-006).S. polyrhiza was authenticated by Professor Hyungwoo Kim.In brief, whole S. polyrhiza (100 g) was extracted, as we previously described [26], and yielded 12.5 g of lyophilized powder (EESP, yield 12.5%).A sample of EESP was deposited in the School of Korean Medicine, Pusan National University (Voucher no.MH2017-006).

Animals
Male Balb/c mice (6 weeks old) were purchased from Samtaco (Osan, Republic of Korea).The mice were housed in a specific pathogen-free environment under a 12 h light/dark cycle and provided standard rodent food and water ad libitum.All animal experiments were conducted in accordance with the guidelines issued by the animal care and use committee of Pusan National University (approval number PNU-2019-2269).

CD Induction and Experimental Schedule
CD was induced using DNFB, as we previously described [26].In brief, mice were randomly divided into six groups, namely, a normal (NOR) group of non-treated mice (n = 6), a control (CTL) group of non-treated CD mice (n = 8), three EESP groups of CD mice treated with 50, 150, or 500 µg/day of EESP for six consecutive days (n = 8/group), and a DEX-treated CD group treated with 150 µg/day of DEX for six consecutive days (n = 8).For CD induction, mice were sensitized by the application of 30 µL DNFB (0.2%, v/v) in acetone: olive oil (AOO, 4:1) onto both ears for three consecutive days.Five days after sensitization, each mouse was challenged by the application of 60 µL DNFB (0.2%, v/v) in AOO onto the shaved back every two days (4 times).EESP and DEX were dissolved in 70% ethanol, diluted in AOO, and then applied onto the shaved backs of mice.The experimental schedule is summarized in the Supplementary Data (Supplementary Data S9, Figure S5).

Skin Surface Scores and Thicknesses Measurements
Skin lesions were imaged using a digital camera (Olympus, Tokyo, Japan), and skin surface scores were evaluated using the modified version of Amano's method [36].In brief, the skin surface score was determined by summing the scores for scaling, excoriation, erythema, and skin roughness assessed using a 4-point scale (0, none; 1, mild; 2, moderate; 3, severe).Skin tissues were cut into 5 mm diameter pieces, and weights were measured using an electronic scale (Sartorius, Seongnam, Republic of Korea).

Erythema and Melanin Indices
Erythema and melanin indices were determined using a dermo-spectrophotometer (Cortex Technology, Hadsund, Denmark) at three different skin locations per mouse.

Histopathological Examinations
Skin tissues were fixed in 10% (v/v) formaldehyde, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin, and histological changes were observed under a light microscope (Carl Zeiss AG, Oberkochen, Germany) at ×50.

Evaluations of Epidermal Hyperplasia and Immune Cell Infiltration
Vertical distances between the basal lamina and outer stratum granulosum were measured to evaluate epidermal hyperplasia.Three random measurements were made per slide using the Zen program (ZEISS, Jena, Germany).Cells were enumerated using a cell-counting grid (2.1 × 1.6 mm, 100× magnification) to evaluate immune cell infiltration in four randomly selected, non-overlapping regions per slide.Immune cells were defined as macrophages, polymorphonuclear leukocytes, lymphocytes, eosinophils, plasma cells, and giant cells [37].The number of infiltrated immune cells was represented as the number per square millimeter.

Statistical Analysis
Data were analysed using One-way ANOVA followed by Dunnett's multiple comparison test.Prime 5 for Windows version 5.01 (GraphPad Software Inc., La Jolla, CA, USA) was used for the analysis.Results are presented as means ± standard deviations (SDs), and statistical significance was accepted for p values < 0.05.

Figure 1 .
Figure 1.Relationship between 29 CD-related targets of S. polyrhiza.The edges indicate both functional and physical protein associations and line thickness indicates the strength of data support.

Figure 1 .
Figure 1.Relationship between 29 CD-related targets of S. polyrhiza.The edges indicate both functional and physical protein associations and line thickness indicates the strength of data support.

Figure 2 .
Figure 2. Correlations between 14 active compounds and the 21 CD-related targets.Green indicates the active compounds, and light blue indicates the target genes.

Figure 2 .
Figure 2. Correlations between 14 active compounds and the 21 CD-related targets.Green indicates the active compounds, and light blue indicates the target genes.

Figure 3 .
Figure 3. Binding modes of the active compounds and residues of two proteins.Eleven active compounds and residues of TNF (A) and eight active compounds and residues of IL-6 (B).Gray cartoons indicate active compounds.

Figure 3 .
Figure 3. Binding modes of the active compounds and residues of two proteins.Eleven active compounds and residues of TNF (A) and eight active compounds and residues of IL-6 (B).Gray cartoons indicate active compounds.

Figure 6 .
Figure 6.Effects of EESP on histopathological abnormalities in inflamed tissues.The presented sequence is the same as that in Figure 5A.The yellow bars indicate epidermal thicknesses (original magnification ×50) (A).Histogram showing mean epidermal thicknesses (B) and numbers of infiltrating immune cells (C) in the six study groups.Abbreviations are as defined in Figure 5. Results are presented as means ± SDs. ### p < 0.001 vs. NOR; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CTL.

Figure 6 .
Figure 6.Effects of EESP on histopathological abnormalities in inflamed tissues.The presented sequence is the same as that in Figure 5A.The yellow bars indicate epidermal thicknesses (original magnification ×50) (A).Histogram showing mean epidermal thicknesses (B) and numbers of infiltrating immune cells (C) in the six study groups.Abbreviations are as defined in Figure 5. Results are presented as means ± SDs. ### p < 0.001 vs. NOR; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CTL.

Figure 8 .
Figure 8. Effects of EESP on DNFB-induced phosphorylation of MAPK signalling pathways in inflamed tissues.MAPK signalling molecules were determined by Western blot.Abbreviations are as defined in Figure 5.

Figure 8 .
Figure 8. Effects of EESP on DNFB-induced phosphorylation of MAPK signalling pathways in inflamed tissues.MAPK signalling molecules were determined by Western blot.Abbreviations are as defined in Figure 5.

Figure 8 .
Figure 8. Effects of EESP on DNFB-induced phosphorylation of MAPK signalling pathways in inflamed tissues.MAPK signalling molecules were determined by Western blot.Abbreviations are as defined in Figure 5.

Table 2 .
Binding affinities for interactions between two proteins (TNF and IL-6) and 14 active compounds.

Table 2 .
Binding affinities for interactions between two proteins (TNF and IL-6) and 14 active compounds.